Enolase activity assay kit

The enolase activity of rCaEno1 was determined using the Enolase Activity Assay Kit (Sigma-Aldrich) according to the manufacturer’s instructions with slight modification. Briefly, the mixture with a final volume of 100 μl (50 μl of sample dilutions + 50 μl of the appropriate reaction mix) was mixed well by pipetting and incubated at 25°C. To test the inhibition of enolase activity by ENOblock, rCaEno1 was preincubated with ENOblock or sodium fluoride (NaF) in a total volume of 50 μl at room temperature, and then the experiment was continued as described above. After 30 min, the absorbance at 570 nm was measured as the OD_{570 nm} value. NaF used as a control is a well-known enolase inhibitor.
The transglutaminase activity of rCaEno1 was determined using Transglutaminase Assay Kit (Sigma) according to the manufacturer’s instructions. The kit can also be used to assay the inhibitory activity of ENOblock. Briefly, rCaEno1 and ENOblock were added into the appropriate wells, and the volume was adjusted to 50 μl with ultrapure water. rCaEno1 was preincubated with ENOblock or cystamine at room temperature, and then the experiment was continued according to the manufacturer’s instructions. The absorbance at 450 nm was measured as the OD_{450 nm} value. Cystamine was used as a control is a specific inhibitor of TGase.

α-enolase enzymatic activity assay was performed using the Enolase Activity Assay Kit from Sigma-Aldrich. First, the reaction mixture was prepared according to the kit protocol. Recombinant enolase was plated in each well with DMSO or 3-HT, and 50 μL of the corresponding reaction mix was added to each well. The reaction was measured at 340 nm over 10 min at 30 s intervals using a multifunctional microplate reader (SpectraMax iD3).

Enolase activity was determined using Enolase Activity Assay Kit according to the manufacturer’s instruction (Sigma, Germany, MAK178-1KT). In brief, the samples of cell lysates were mixed with reaction buffer and incubated at 25 °C. After 5–10 min, an initial measurement of OD value was taken at a wavelength of 570 nm, and was followed by measurement every 2–3 min until the OD value of the most active sample was greater than the value of the highest standard for getting the final measurement. Enolase activity was calculated using an equation described previously^{40 (link)}.